We have been interested in the mechanism of action of chaperone proteins in protein folding. We have been using an in vitro replication system that replicates DNA carrying the plasmid P1 origin of replication as a model system to study the function of three E. coli heat shock proteins, DnaJ, DnaK (the Hsp70 homolog), and GrpE. We found that DnaJ and DnaK, in an ATP-dependent reaction, activate the sequence specific DNA binding of the P1 initiator protein, RepA. We discovered that activation converts RepA dimers to monomers and that the monomer form binds with high affinity to the P1 origin DNA. The enzymatic activation can be mimicked by converting RepA dimers to monomers by urea denaturation followed by renaturation. We found that urea-treated repA bypasses the requirement for DnaJ, DnaK, and GrpE in in vitro complementation assays with crude extracts of dnaJ, dnaK, and grpE mutant cells without the addition of purified DnaJ, DnaK, or GrpE, respectively. Thus, DnaJ, DnaK, and GrpE are required exclusively for the activation of RepA for P1 plasmid DNA synthesis in vitro.